Identification of important interactions between subchondral bone ...

CHAPTER 7: PAPER IV
The pre-stimulated cartilage incubated in MMP buffer increased the levels of CTX-II, and
AGNx-II, suggesting aggrecanases and MMPs to be the key proteases to generate these two
biomarkers. The MMP inhibitor, GM6001, decreased the increased levels of the two biomarkers
in both 4 and 11 days pre-stimulated cartilage explants, which correlates with the literature that
GM6001 inhibits both aggrecanases and MMPs 11 . The biomarker levels varied significantly
between the normal, cytokine-stimulated and OA cartilage explants, showing that the pre-
stimulated bovine cartilage explants did not represent human OA cartilage. However, the results
informed us instead that the presence and role of endogenous proteases vary from healthy
cartilage and during the progression of OA. Thus it is important to target the right proteases at
the right time, to receive the optimal treatment.
E64 indicated that cysteine proteases mask the levels of the biomarker representing
MMP-mediated aggrecan degradation, whereas GM6001 indicated that MMPs masks the levels of
the collagen type II degradation biomarkers in human OA (Table1/2). Sondergaard et al. 12 have
shown that CTX-II levels increases in the presence of E64 in bovine cartilage explants (living
tissue), suggesting that it is a compensatory degrading effect from other proteases, like MMPs,
when cysteine proteases are absent to degrade the proteins. However, it could also be a masking
effect from the cysteine proteases that cleaves MMP-mediated CTX-II fragments further, and
thus masks the levels, which is actually degraded by the MMPs. Our study confirms that the study
by Sondergaard et al. 12 must have been a compensatory effect, as we did not see any increase of
CTX-II by E64 in cytokine-stimulated bovine explants, in which the chondrocytes were
inactivated. If E64 should have conducted a masking effect in the Sondergaard 12 study, our E64
should have been increased as well.
The present brief report will be followed by a thorough study with regards to the
dynamics of proteases in diseased and normal cartilage, and potential compensatory effects of the
different proteases herein. A complete understanding of the common molecular sequence of
events underlying the development of OA will expand our knowledge of the pathogenesis of
OA. These events will also contribute with important information to search for novel therapeutic
targets for the prevention and treatment of OA at the right time during the progression of the
disease.
Conclusions
Taken together, these experiments suggest that CTX-II, NBC2 and AGNx-II are released in part
by different proteolytic pathways in human OA cartilage. Furthermore, the presence and role of
endogenous proteases probably depend on the stage of OA, as we did not observe the same
release of biomarkers during inhibition of one type of protease, in normal versus cytokine-
stimulated and OA cartilage. Thus more investigation on this topic may have significant value for
development of new treatments for OA.
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